Sirtuin 3 improves fatty acid metabolism in response to high nonesterified fatty acids in calf hepatocytes by modulating gene expression

Abstract

Sirtuin 3 (SIRT3), a mitochondrial deacetylase, is a key regulator of energy metabolism in the liver. In nonruminants, the hepatic abundance of SIRT3 is decreased in individuals with nonalcoholic fatty liver diseases, and recovery of SIRT3 alleviates hepatic triacylglycerol (TG) deposition. However, the level of SIRT3 expression and its effects on lipid metabolism in dairy cows have not been characterized. Here we studied the hepatic expression of SIRT3 in cows with fatty liver and the role of SIRT3 in fatty acid metabolism in bovine hepatocytes. This in vivo study involved 10 healthy cows and 10 cows with fatty liver, from which we collected samples of liver tissue and blood. Primary hepatocytes were isolated from Holstein calves and treated with 0, 0.5, or 1.0 mM nonesterified fatty acids (NEFA) for 24 h or transinfected with SIRT3 overexpression adenovirus (Ad-SIRT3)/SIRT3-short interfering (si)RNA for 48 h. Cows with fatty liver displayed lower serum glucose concentrations but higher serum NEFA and β-hydroxybutyrate concentrations relative to healthy cows. Cows with fatty liver also had significant lower mRNA and protein abundance of hepatic SIRT3. Incubation of primary hepatocytes with NEFA reduced SIRT3 abundance in primary hepatocytes in a dose-dependent manner. Fatty acid (1 mM) treatment also markedly increased the abundance of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FAS) but significantly decreased the abundance of carnitine palmitoyltransferase I (CPT1A), carnitine palmitoyltransferase II (CPT2), and acyl-CoA oxidase (ACO). Knockdown of SIRT3 by SIRT3-siRNA downregulated the mRNA abundance of CPT1A, CPT2, and ACO. In contrast, overexpression of SIRT3 by Ad-SIRT3 upregulated the mRNA abundance of CPT1A, CPT2, and ACO; downregulated the mRNA abundance of ACC1 and FAS; and consequently, decreased intracellular TG concentrations. Overexpression of SIRT3 ameliorated exogenous NEFA-induced TG accumulation by downregulating the abundance of ACC1 and FAS and upregulating the abundance of CPT1A, CPT2, and ACO in calf hepatocytes. Our data demonstrated that cows with fatty liver had lower hepatic SIRT3 contents, and an increase in SIRT3 abundance by overexpression mitigated TG deposition by modulating the expression of lipid metabolism genes in bovine hepatocytes. These data suggest a possible role of SIRT3 as a therapeutic target for fatty liver disease prevention in periparturient dairy cattle.

Sirtuin 3 Restores Synthesis and Secretion of Very Low-Density Lipoproteins in Cow Hepatocytes Challenged with Nonesterified Fatty Acids In Vitro

“…The empty adenovirus vector (Ad-green fluorescent protein, Ad-GFP), SIRT3 overexpression adenovirus (Ad-SIRT3), and short interfering RNA (siRNA) were constructed by Hanbio (Shanghai, China) as described elsewhere [ 13 ]. Briefly, the cDNA sequence of bovine SIRT3 was synthesized and further cloned into pHBAd-EF1a-MCS-3flag-CMV-EGFP vectors.…”

“…We followed a similar protocol for the isolation and culture of cow hepatocytes as in our previous study [ 13 ]. The protocol was approved by the Institutional Animal Care and Use Committee of Hunan Agricultural University (Changsha, China).…”

“…Sirtuins 3 (SIRT3), a type of nicotinamide adenine dinucleotide (NAD + )-dependent deacetylases, regulates cellular energy metabolism and redox homeostasis in nonruminants [ 11 ]. Hepatic SIRT3 expression decreased in the first two weeks after calving in dairy goats [ 12 ]; hepatic SIRT3 content was lower in cows with fatty liver compared to healthy controls [ 13 ]. Additionally, overexpression of SIRT3 alleviates TAG deposition induced by high NEFA via inhibition of fatty acid synthesis and promotion of fatty acid oxidation in cow hepatocytes [ 13 ].…”

“…Hepatic SIRT3 expression decreased in the first two weeks after calving in dairy goats [ 12 ]; hepatic SIRT3 content was lower in cows with fatty liver compared to healthy controls [ 13 ]. Additionally, overexpression of SIRT3 alleviates TAG deposition induced by high NEFA via inhibition of fatty acid synthesis and promotion of fatty acid oxidation in cow hepatocytes [ 13 ]. Accumulating evidence shows that SIRT3 may serve as an antagonist against the pathophysiological events induced by high NEFA [ 12 , 13 ].…”

“…Additionally, overexpression of SIRT3 alleviates TAG deposition induced by high NEFA via inhibition of fatty acid synthesis and promotion of fatty acid oxidation in cow hepatocytes [ 13 ]. Accumulating evidence shows that SIRT3 may serve as an antagonist against the pathophysiological events induced by high NEFA [ 12 , 13 ]. Thus far, the role of SIRT3 in the VLDL synthesis and assembly has not been investigated extensively.…”